Carrageenophyte identification by second-derivative Fourier transform infrared spectroscopy

The second-derivative mode of the Fourier transform I.R. spectra of dried algal material has been applied to distinguish the carrageenans-producingStenogramme interrupta from the isomorphous speciesRhodymenia howeana. Spectra of the tetrasporophyteS. interrupta showed bands assigned to a -carrageenan type polysaccharide, while the gametophytic and cystocarpic plants showed the characteristic absorptions of -and -carrageenans. Results were confirmed by hot water extraction of samples of the three nuclear phases ofS. interrupta and characterization of the extracts by chemical analysis.Author for correspondence     

Membrane-induced peptide structural changes monitored by infrared and circular dichroism spectroscopy

As more peptide secondary structures deduced by infrared spectroscopy (IR) have been reported in the literature, there have been overlaps in assignments of elements of secondary structure to carbonyl vibrational frequencies. We have investigated this phenomenon with regards to the use of IR for monitoring membrane-induced structural changes using conformationally diverse peptides. These IR studies, complemented by circular dichroism (CD) experiments, revealed that peptide–solvent interactions can mask membrane-induced conformational changes monitored by IR. A structural transition from random coil to α-helix upon the binding of mastoparan X to a membrane was clearly observed by CD but obscured in the amide I region of the IR spectrum. In addition, unlike the buried helical peptides gramicidin D and P16 in micelles, the amide II peak for mastoparan X was absent, likely due to H–D exchange. This suggests information on the peptide's membrane-bound solvent accessibility could be obtained from this region of the spectrum.     

Infrared studies of water induced conformational changes in bacteriorhodopsin

Fourier transform infrared difference spectroscopy has been used to study the effect of water on the conformation of bacteriorhodopsin. The infrared spectra as a function of water content show a conformational change at about 0.06 g H₂O/g bacteriorhodopsin. By an interference method the thickness of the sample was measured and shows similar behavior as a function of water content. This study gives insight into the process of water absorption by purple membrane. The observations are in good agreement with those found for other proteins.Abbreviations IR infrared - FTIR Fourier transform IR     

Probing conformational changes in the nicotinic acetylcholine receptor by Fourier transform infrared difference spectroscopy.

We have developed a Fourier transform infrared (FTIR) difference method for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR). Our approach is to deposit reconstituted nAChR membranes in a thin film on the surface of a germanium internal reflection element, acquire FTIR spectra in the presence of bulk aqueous solution using attenuated total reflection, and then trigger conformational changes by sequentially flowing a buffer either with or without an agonist past the film surface. Using the fluorescent probe, ethidium bromide, it is demonstrated that the method of nAChR film deposition does not affect the ability of the receptor to undergo the resting-to-desensitized state transition. The difference of FTIR spectra of nAChR films recorded in the presence and absence of agonists reveal highly reproducible infrared bands that are not observed in the difference of spectra recorded with only buffer flowing past the film surface. Some of the bands are assigned to changes in protein secondary structure and to changes in the structure of individual amino acid residues. Bands arising from the vibrations of the agonist bound to the receptor are also observed. The results demonstrate that FTIR difference spectroscopy can detect structural changes in the nAChR that occur upon the binding of ligands. The technique will be an effective method for investigating nAChR structure and function as well as receptor-drug interactions.     

Probing the conformational changes and peroxidase-like activity of cytochrome c upon interaction with iron nanoparticles

Herein, the interaction of iron nanoparticle (Fe-NP) with cytochrome c (Cyt c) was investigated, and a range of techniques such as dynamic light scattering (DLS), zeta potential measurements, static and synchronous fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and ultraviolet–visible (UV–vis) spectroscopy were used to analyze the interaction between Cyt c and Fe-NP. DLS and zeta potential measurements showed that the values of hydrodynamic radius and charge distribution of Fe-NP are 83.95 ± 3.7 nm and 4.5 ± .8 mV, respectively. The fluorescence spectroscopy results demonstrated that the binding of Fe-NP with Cyt c is mediated by hydrogen bonds and van der Waals interactions. Also Fe-NP induced conformational changes in Cyt c and reduced the melting temperature value of Cyt c from 79.18 to 71.33°C. CD experiments of interaction between Fe-NP and Cyt c revealed that the secondary structure of Cyt c with the dominant α-helix structures remained unchanged whereas the tertiary structure and heme position of Cyt c are subjected to remarkable changes. Absorption spectroscopy at 695 nm revealed that Fe-NP considerably disrupt the Fe…S(Met80) bond. In addition, the UV–vis experiment showed the peroxidase-like activity of Cyt c upon interaction with Fe-NP. Hence, the data indicate the Fe-NP results in unfolding of Cyt c and subsequent peroxidase-like activity of denatured species. It was concluded that a comprehensive study of the interaction of Fe-NP with biological system is a crucial step for their potential application as intracellular delivery carriers and medicinal agents.     

Evaluation of infrared spectroscopy as a bacterial identification method

Summary A study motivated by the recent revival of interest in the use of IR spectroscopy to identify bacteria is reported. A library of FT-IR spectra of dried bacterial films was compled using 16 different strains. A test set was complied from spectra of the same strains grown several months later. The test set was quantitatively compared with the library on the basis of spectral similarity in the region 980–1190 cm^–1. Six of the strains in the test set were not matched with the correct strain in the library despite efforts to reproduce the conditions under which cells were grown and prepared. The results suggest that reproducibility of the bacterial spectra is a potential difficulty that must be addressed by any attempts to develop FT-IR spectroscopy as a bacterial identification method.     

Effects of zinc on creatine kinase: activity changes, conformational changes, and aggregation

The effects of zinc on creatine kinase (CK) are very distinctive compared with other bivalent metal ions. Zinc up to 0.1 mM induced increases in CK activity, accompanied by significant hydrophobic surface exposure and increase in a-helix content of CK. Zinc over 0.1 mM denatured and inactived CK. In the presence of 0.1 mM zinc, the CK activity was very close to that of the native CK, but its conformation changed greatly. The kinetic courses of CK inactivation and conformational change in the presence of 1 mM zinc were measured to determine apparent rate constants of inactivation and conformational change. Zinc over 0.05 mM induced CK aggregation at 37°C, and the aggregation was dependent on zinc concentration, CK concentration, and temperature. The inactivation and aggregation can be reversed by EDTA. An explanation for CK aggregation induced by zinc is proposed, as well as a mechanism for CK abnormality in Alzheimer's disease.To whom correspondence should be addressed.     

Probing protonation/deprotonation of tyrosine residues in cytochrome ba3 oxidase from Thermus thermophilus by time-resolved step-scan Fourier transform infrared spectroscopy

Elucidating the properties of the heme Fe-Cu(B) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν(7a)(CO) of the protonated form of Tyr residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). By monitoring the intensity changes of the 1247 and 1301 cm(-1) modes as a function of pH, we measured a pK(a) of 7.8 for the observed tyrosine. The FTIR spectral changes associated with the tyrosine do not belong to Tyr-237 but are attributed to the highly conserved in heme-copper oxidases Tyr-136 and/or Tyr-133 residue (Koutsoupakis, K., Stavrakis, S., Pinakoulaki, E., Soulimane, T., and Varotsis, C. (2002) J. Biol. Chem. 277, 32860-32866). The oxygenation of CO by the mixed-valence form of the enzyme revealed the formation of the ～607 nm P (Fe(IV)=O) species in the pH 6-9 range and the return to the oxidized form without the formation of the 580 nm F form. The data indicate that Tyr-237 is not involved in the proton transfer pathway in the oxygenation of CO by the mixed-valence form of the enzyme. The implication of these results with respect to the role of Tyr-136 and Tyr-133 in proton transfer/gating along with heme a(3) ring D propionate-H(2)O-ring A propionate-Asp-372 site to the exit/output proton channel (H(2)O pool) is discussed.     

Water near lipid membranes as seen by infrared spectroscopy

The ordering and H-bonding characteristics of the hydration water of the lipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were studied using polarized infrared spectroscopy by varying either the temperature or the relative humidity of the ambient atmosphere of multibilayer samples. The OH-stretching band of lipid-bound water was interpreted by a simplified two-state model of well-structured, low density “network” water and of less-structured dense “multimer” water. The IR-spectroscopic data reflect a rather continuous change of the water properties with increasing distance from the membrane and with changing temperature. Network and multimer water distribute across the whole polar interphase with changing composition and orientation. Upon dehydration the fraction of network water increases from about 30 to 60%, a value which is similar to that in supercooled water at −25°C. The highly ordered gel phase gives rise to an increased fraction of structured network water compared with the liquid crystalline phase. The IR order parameter shows that the water dipoles rearrange from a more parallel towards a more perpendicular orientation with respect to the membrane normal with progressive hydration. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Application of computerized infrared and Raman spectroscopy to conformation studies of casein and other food proteins

Summary The ability of modern biotechnology to produce new or modified proteins has outpaced current understanding of the relationship between protein structure and protein function. Resolution-enhanced infrared spectroscopy and Raman spectroscopy are excellent non-destructive techniques for investigating the secondary structure of proteins under a wide variety of conditions. The techniques yield rapid, reliable estimates of the proportion of helical structure, -strands, and turns of proteins in solution, as gels, or as solids. These methodologies can also detect subtle variations in protein conformation that frequently occur upon change of the biomolecular environment. In particular, it is possible to study structural changes which arise from alterations in pH, ionic strength, nature of solvent, and from interactions with other molecules or ions, such as another protein or Ca²⁺ ions. The first part of this paper will briefly review various important aspects of the techniques. The subsequent part describes application to structural problems of casein and other food proteins.     

Comparative study of non-invasive monitoring via infrared spectroscopy for mammalian cell cultivations

Process analytical technology (PAT) is a guide to improve process development in biotech industry. Optical sensors such as near and mid infrared spectrometers fulfill an essential part for PAT. NIRS and MIRS were investigated as non-invasive on line monitoring tools for animal cell cultivations in order to predict critical process parameters, like cell parameters as well as substrate and metabolite concentrations. Eight cultivations were performed with frequent sampling. Variances between cultivations were induced by spiking experiments with intent to break correlations between analytes; to keep causality of the models; and to increase model robustness.     

Detection of apolipoprotein B100 early conformational changes during oxidation

Conformational changes of human plasma apolipoprotein B100 (apoB) during oxidative modification of low-density lipoproteins (LDL) have been investigated. Emphasis has been put on the early stages of LDL oxidation and the modification of apoB. We have applied two different modes of LDL oxidation initiation in order to approach the problem from different perspectives. To study conformational changes of the protein and the phospholipids surface monolayer, we have applied attenuated total reflection infrared as well as fluorescence spectroscopy. We have found for the first time that conformational changes of apoB occur even in the earliest stages of oxidation process and that those are located predominantly in the β-sheet regions. The dynamics of changes has also been described and related to different stages of oxidation. After initial increase in particle surface accessibility and mobility, by entering into the propagation phase of oxidation process, LDL surface accessibility and mobility are decreased. Finally, in the decomposition phase of LDL oxidation, as the particle faces large chemical and physical changes, surface mobility and accessibility is increased again. These observations provide new insights into the modifications of LDL particles upon oxidation.     

Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.     

Orientation of intrinsic proteins in photosynthetic membranes. Polarized infrared spectroscopy of chloroplasts and chromatophores

In order to estimate the degree of orientation of the α-helices of intrinsic proteins in photosynthetic membranes, polarized infrared spectroscopy has been used to measure the dichroism of the amide I and amide II absorption bands of air-dried oriented samples of purple membranes, chloroplasts and chromatophores from Rhodopseudomonas sphaeroides. Using purple membrane, in which the orientation of the α-helices is precisely known (Henderson, R. (1977) Annu. Rev. Biophys. Bioeng. 6, 87–109), as a standard to calibrate our measurements and estimating the mosaic spread (extent of orientation) of the membranes from linear dichroism measurements performed in the visible spectral range, it is concluded that in photosynthetic membranes, the α-helices of intrinsic proteins are tilted at less than 40° with respect to the normal to the plane of the membrane.     

An alternative infrared spectroscopy assay for the quantification of polysaccharides in bacterial samples

The ability of bacteria to produce extracellular polysaccharides has been regarded as an indication of biofilm-forming capacity. Therefore, the determination of the sugar content in bacterial samples becomes a significant parameter. The colorimetric methods currently used are rather sensitive to the nature of the sugars and therefore require knowledge of the sugar types present in the samples. Unfortunately, the types of sugars present in bacteria are generally unknown and often composed of a complex mixture. In this article, we propose an alternative method based on Fourier transform infrared (FTIR) spectroscopy for the estimation of the total sugar content in bacterial samples. The method is based on a systematic treatment of FTIR spectra obtained from dried bacteria samples. It is assumed that the total sugar amount can be estimated from the area of characteristic bands between 970 and 1182 cm(-1). In parallel, the amide II band (1560-1530 cm(-1)) associated with proteins, or the C-H stretching region (2820-3020 cm(-1)) associated with the biomass, can be used for normalization purposes. Therefore, the ratio of the band area in the sugar window over that of the amide II or C-H stretching can be used to report the sugar content in bacterial samples. This method has been validated on model bacterial mixtures containing sugars, proteins, and DNA. Results with real bacterial samples are also provided and show conclusively that increased sugar contents in biofilms can be identified. The proposed FTIR approach requires minimal sample preparation and a single acquisition, is rapid, and may be applied to any kind of bacterial growth.     

Substrate-induced conformational changes of melibiose permease from Escherichia coli studied by infrared difference spectroscopy

Fourier transform infrared difference spectroscopy has been used to obtain information about substrate-induced structural changes of the melibiose permease (MelB) from Escherichia coli reconstituted into liposomes. Binding of the cosubstrate Na(+) gives rise to several peaks in the amide I and II regions of the difference spectrum Na(+).MelB minus H(+).MelB, that denote the presence of conformational changes in all types of secondary structures (alpha-helices, beta-sheets, loops). In addition, peaks around 1400 and at 1740-1720 cm(-1) are indicative of changes in protonation/deprotonation or in environment of carboxylic groups. Binding of the cosubstrate Li(+) produces a difference spectrum that is also indicative of conformational changes, but that is at variance as compared to that induced by Na(+) binding. To analyze the following transport steps, the melibiose permease with either H(+), Na(+), or Li(+) bound was incubated with melibiose. The difference spectra obtained by subtracting the spectrum cation.MelB from the respective complex cation.melibiose.MelB were roughly similar among them, but different from those induced by cation binding, and more intense. Therefore, major conformational changes that are induced during melibiose binding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm(-)(1), are similar for the three cotransporting cations. Changes in the protonation state and/or in the environment of given carboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.     

Assessment of temperature effects on beta-aggregation of native and glycated albumin by FTIR spectroscopy and PAGE: relations between structural changes and antioxidant properties

Structural modifications of bovine serum albumin (BSA) induced by heating, and the involvement of glycation of albumin in such processing were studied by using Fourier transform infrared spectroscopy (FTIR) and polyacrylamide gel electrophoresis (PAGE). For native BSA, heating treatments gave rise to beta structures which were amplified to the detriment of alpha-helix form, and which were associated with increased aggregation. A very high correlation was obtained between FTIR Amide I band evolution and aggregation rate parameters, showing the contribution of beta-form in aggregates formation. We further assessed the effect of glycation on protein sensibility to heating treatments. A reduction of conformational changes and aggregation processes was demonstrated for the glycated form of the protein. The antioxidant properties of albumin were evaluated using two different techniques assessing metal binding and free radical neutralizing capacities of the protein. Associations between structural changes in BSA induced by the thermal treatment and its antioxidant activities were established.     

Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface

We have shown that the molecular conformation of a protein at an interface can be probed spatially using time-resolved evanescent wave-induced fluorescence spectroscopic (TREWIFS) techniques. Specifically, by varying the penetration depth of the evanescent field, variable-angle TREWIFS, coupled with variable-angle evanescent wave-induced time-resolved fluorescence anisotropy measurements, allow us to monitor how fluorescence intensity and fluorescence depolarization vary normal to an interface as a function of time after excitation. We have applied this technique to the study of bovine serum albumin (BSA) complexed noncovalently with the fluorophore 1-anilinonaphthalene-8-sulfonic acid. The fluorescence decay varies as a function of the penetration depth of the evanescent wave in a manner that indicates a gradient of hydrophobicity through the adsorbed protein, normal to the interface. Restriction of the fluorescent probes motion also occurs as a function of distance normal to the interface. The results are consistent with a model of partial protein denaturation: at the surface, an adsorbed BSA molecule unfolds, thus optimizing protein–silica interactions and the number of points of attachment to the surface. Further away, normal to the surface, the protein molecule maintains its coiled structure.Submitted as a record of the 2002 Australian Biophysical Society meeting